Beta-defensins

ABSTRACT

The present invention relates to polynucleotide and polypeptide molecules for zamp1, a novel member of the β-defensin family. The polypeptides, and polynucleotides encoding them, exhibit anti-microbial activity and may be used in the study or treatment of microbial infections. The present invention also includes antibodies to the zamp1 polypeptides.

REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.09/636,399, filed on Aug. 10, 2000, now U.S. Pat. No. 6,576,755, whichis a continuation-in-part of U.S. patent application Ser. No.09/344,097, filed on Jun. 25, 1999 now abondoned, which is acontinuation-in-part of U.S. patent application Ser. No. 09/150,786,filed on Sep. 10, 1998 now abandoned , which claims the benefit of U.S.Provisional Application Ser. No. 60/058,335, filed on Sep. 10, 1997 andU.S. Provisional Application Ser. No. 60/064,294, filed on Nov. 5, 1997,all of which are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Biological defense strategies have evolved to protect organisms frominvasion by other species. Microbial infection response systems includeoxidative and non-oxidative mechanisms, utilizing compounds that areenzymatically synthesized in cells and peptides that are single geneproducts.

Anti-microbial peptides constitute an oxygen-independent host defensesystem found in organisms encompassing many taxonomic families. Onemajor class of anti-microbial peptides can be sequence-defined byconserved cysteine residue patterns and are termed defensins. Mammaliandefensins, derived from skin, lung and intestine, exhibit antibioticactivity against a wide variety of pathogens, including gram-positiveand gram-negative bacteria, fungi (e.g. Candida species) and viruses.See, for example, Porter et al., Infect. Immun. 65(6): 2396–401, 1997.

The amphipathic character of the defensin peptides appears to be the keyto the general mechanism of microbial attack, i.e., by creating pores,or “boring” through the cell wall. In addition, Daher et al., J. Virol.60(3): 1068–74, 1986, reported that enveloped viruses, including herpessimplex types 1 and 2, cytomegalovirus and influenza virus (A/WSN),among others, were inactivated by incubation with human neutrophilpeptide (HNP-1) and speculated that the binding of defensin molecules toviruses impairs the virus' ability to infect cells.

The defensin family of anti-microbial peptides can be divided into twomajor subclasses based on two distinct consensus sequences. See, forexample, Martin et al., Journal of Leukocyte Biology 58: 128–36, 1995.The first defensin subclass, classic defensins, represented by HNPs arestored in the so-called large azurophil granules of neutrophils andmacrophages and attack microorganisms that have been phagocytosed bythese cells. The amino acid sequence of HNPs is consistent with apredicted disulfide bridging that is distinct from that of theβ-defensin subclass. Epithelial cells can also be a source of defensins,and these cells appear to secrete these peptides into the external,extra-cellular environment. In the mouse, for example, Paneth cells ofthe small intestine and proximal colon, secrete defensin-like peptides,called cryptidins, into the lumen. See, for example, Ouellette andSelsted, The FASEB Journal 10: 1280–9, 1996.

β-defensins, the second major defensin subclass, include peptides foundin bovine lung (e.g., BNBD-bovine neutrophil β-defensins) as well as asecreted form (TAP—tracheal anti-microbial peptide). See, for example,Selsted et al., J. Biol. Chem. 268(9): 6641–8, 1993. Two humanβ-defensins have been reported. SAP-1 was isolated from human psoriaticskin, and hBD-1 was found in low concentrations in human blood filtrate.See, for example, Bensch et al., FEBS Lett. 368(2): 331–5, 1995). Theamino acid sequence of these human β-defensins is most similar to thebovine BNDPs and TAP. See, for example, Harder et al., Nature 387: 861,1997, wherein SAP-1 is designated hBD-2.

Other than the conserved cysteine residues the defensin family is quitesequence divergent. It is possible that the variant amino acid positionsmay be related to the site or conditions of activity or to the spectrumof pathogens attacked by a particular defensin.

In addition to anti-microbial activities, particular defensins exhibitmetabolically sensitive cytotoxic activity (Lichtenstein et al., Blood68: 1407–10, 1986 and Sheu et al., Antimicrob. Agents Chemother. 28:626–9, 1993), alter the response of adrenal cortical cells to ACTH (Zhuet al., Proc. Natl. Acad. Sci. (USA) 85(2): 592–6, 1988) and havespecific chemotactic activity for human monocytes (Territo et al., J.Clin. Invest. 84(6): 2017–20, 1989). Recruitment of monocytes byneutrophils may, in part, be mediated by neutrophilic defensins andsuggests a pro-inflammatory activity for these peptides in addition totheir anti-microbial effects. Also, a decrease in defensin mRNA levelhas been demonstrated in SPG (specific granule disease). See, forexample, Tamura et al., Japan. Int. J. Hematol. 59(2): 137–42, 1994.Higazi et al., J. Biol. Chem. 271(3): 17650–5, 1996, suggested thatplasminogen bound to fibrin in the presence of defensin may be lesssusceptible to activation by tPA.

Moieties having anti-microbial, immunostimulatory, pro-inflammatory andother properties of defensins are sought. The present invention providessuch polypeptides for these and other uses that should be apparent tothose skilled in the art from the teachings herein.

SUMMARY OF THE INVENTION

Within one aspect the invention provides an isolated protein comprisinga polypeptide that is at least 80% identical to a polypeptide selectedfrom the group consisting of: a) a polypeptide having the sequence ofamino acid residue 1 to amino acid residue 65 of SEQ ID NO:2; b) apolypeptide having the sequence of amino acid residue 19 to amino acidresidue 65 of SEQ ID NO:2; c) a polypeptide having the sequence of aminoacid residue 21 to amino acid residue 65 of SEQ ID NO:2; d) apolypeptide having the sequence of amino acid residue 1 to amino acidresidue 67 of SEQ ID NO:10; e) a polypeptide having the sequence ofamino acid residue 21 to amino acid residue 67 of SEQ ID NO:10; and f) apolypeptide having the sequence of amino acid residue 23 to amino acidresidue 67 of SEQ ID NO:10; wherein the polypeptide has cysteineresidues corresponding to amino acid residues 33, 40, 45, 55, 62 and 63of SEQ ID NOs:2 or 10. Within one embodiment the protein comprises apolypeptide having the sequence selected from the group consisting of:a) a polypeptide having the sequence of amino acid residue 1 to aminoacid residue 67 of SEQ ID NO:10; b) a polypeptide having the sequence ofamino acid residue 21 to amino acid residue 67 of SEQ ID NO:10; and c) apolypeptide having the sequence of amino acid residue 23 to amino acidresidue 67 of SEQ ID NO:10.

Within another aspect is provided an isolated protein having thesequence of SEQ ID NO:10 from amino acid residue 23 to amino acidresidue 67.

Within another aspect the invention provides a polypeptide selected fromthe group consisting of: a) amino acid residue 30 to amino acid residue63 of SEQ ID NO:2; b) amino acid residue 31 to amino acid residue 63 ofSEQ ID NO:2; c) amino acid residue 30 to amino acid residue 64 of SEQ IDNO:2; d) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2;and e) a polypeptide chosen from SEQ ID NOs:14–72.

Within still another aspect is provided a pharmaceutical compositioncomprising a polypeptide selected from the group consisting of: a) aprotein according to claim 1, b) amino acid residue 30 to amino acidresidue 63 of SEQ ID NO:2; c) amino acid residue 31 to amino acidresidue 63 of SEQ ID NO:2; d) amino acid residue 30 to amino acidresidue 64 of SEQ ID NO:2; e) amino acid residue 31 to amino acidresidue 64 of SEQ ID NO:2; and f) a polypeptide chosen from SEQ IDNOs:14–72; in combination with a pharmaceutically acceptable vehicle.

Within yet another aspect is provided an antibody that specificallybinds to a protein as described above.

Within a further aspect is provided an anti-idiotypic antibody of anantibody which specifically binds to a protein as described above.

Within another aspect is provided an isolated polynucleotide moleculeencoding a protein, the polynucleotide molecule consisting of a codingstrand and a complementary non-coding strand, wherein the polynucleotidemolecule encodes a polypeptide that is at least 80% identical to theamino acid sequence to a polypeptide selected from the group consistingof: a) a polypeptide having the sequence of amino acid residue 1 toamino acid residue 65 of SEQ ID NO:2; b) a polypeptide having thesequence of amino acid residue 19 to amino acid residue 65 of SEQ IDNO:2; c) a polypeptide having the sequence of amino acid residue 21 toamino acid residue 65 of SEQ ID NO: 2; d) a polypeptide having thesequence of amino acid residue 1 to amino acid residue 67 of SEQ IDNO:10; e) a polypeptide having the sequence of amino acid residue 21 toamino acid residue 67 of SEQ ID NO:10; and f) a polypeptide having thesequence of amino acid residue 23 to amino acid residue 67 of SEQ IDNO:10; wherein the polypeptide has cysteine residues corresponding toamino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NOs:2 or 10.

Within another aspect the invention provides an isolated polynucleotidemolecule encoding a protein having cysteine residues corresponding toamino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NO:10, thepolynucleotide molecule consisting of a coding strand and acomplementary non-coding strand, wherein the polynucleotide comprises anucleotide sequence that is at least 80% identical to the sequence of apolynucleotide selected from the group consisting of: a) apolynucleotide as shown in SEQ ID NO:9 from nucleotide 220 to nucleotide420; b) a polynucleotide as shown in SEQ ID NO:9 from nucleotide 280 tonucleotide 420; and c) a polynucleotide as shown in SEQ ID NO:9 fromnucleotide 286 to nucleotide 420.

Within yet another aspect is provided an isolated polynucleotidemolecule encoding a protein having cysteine residues corresponding toamino acid residues 33, 40, 45, 55, 62 and 63 of SEQ ID NO:10, thepolynucleotide molecule consisting of a coding strand and acomplementary non-coding strand, wherein the polynucleotide comprises anucleotide sequence as shown in SEQ ID NO:11.

Within another aspect is provided an isolated polynucleotide moleculeencoding a polypeptide selected from the group consisting of: a) aminoacid residue 30 to amino acid residue 63 of SEQ ID NO:2; b) amino acidresidue 31 to amino acid residue 63 of SEQ ID NO:2; c) amino acidresidue 30 to amino acid residue 64 of SEQ ID NO:2; d) amino acidresidue 31 to amino acid residue 64 of SEQ ID NO:2; and e) a polypeptidechosen from SEQ ID NOs:14–72.

The invention also provides an isolated polynucleotide molecule selectedfrom the group consisting of: a) nucleotide 88 to nucleotide 189 of SEQID NO:1; b) nucleotide 88 to nucleotide 192 of SEQ ID NO:1; c)nucleotide 91 to nucleotide 189 of SEQ ID NO:1; d) nucleotide 91 tonucleotide 192 of SEQ ID NO:1; e) nucleotide 88 to nucleotide 189 of SEQID NO:4; f) nucleotide 88 to nucleotide 192 of SEQ ID NO:4; g)nucleotide 91 to nucleotide 189 of SEQ ID NO:4 and h) nucleotide 91 tonucleotide 192 of SEQ ID NO:4.

Within still another aspect is provided an expression vector comprisingthe following operably linked elements: a transcription promoter; a DNAsegment encoding a polypeptide selected from the group consisting of: a)a protein of claim 1; a) amino acid residue 30 to amino acid residue 63of SEQ ID NO:2; b) amino acid residue 31 to amino acid residue 63 of SEQID NO:2; c) amino acid residue 30 to amino acid residue 64 of SEQ IDNO:2; d) amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2;and e) a polypeptide chosen from SEQ ID NOs:14–72; and a transcriptionterminator. Within one embodiment the DNA segment further encodes asecretory signal sequence operably linked to the protein. Within arelated embodiment the secretory signal sequence is selected from thegroup consisting of: a) a polypeptide having the sequence of amino acidresidue 1 to amino acid residue 18 of SEQ ID NO:2; b) a polypeptidehaving the sequence of amino acid residue 1 to amino acid residue 20 ofSEQ ID NO:2; c) a polypeptide having the sequence of amino acid residue1 to amino acid residue 20 of SEQ ID NO:10; and d) a polypeptide havingthe sequence of amino acid residue 1 to amino acid residue 22 of SEQ IDNO:10.

Within another aspect the invention provides a cultured cell into whichhas been introduced an expression vector comprising the followingoperably linked elements: a transcription promoter; a DNA segmentencoding a polypeptide as described above; and a transcriptionterminator; wherein the cell expresses the protein encoded by the DNAsegment.

Within a further aspect is provided a method of producing a proteincomprising: culturing a cell into which has been introduced anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide asdescribed above; and a transcription terminator; whereby the cellexpresses the protein encoded by the DNA segment; and recovering theexpressed protein.

The invention also provides an oligonucleotide probe or primercomprising at least 14 contiguous nucleotides of a polynucleotide of SEQID NO:11 or a sequence complementary to SEQ ID NO:11.

The invention further provides a method of treating a microbial-relateddisease comprising administering to a mammal a therapeutically effectiveamount of a polypeptide selected from the group consisting of: a) apolypeptide of SEQ ID NO:2; b) a polypeptide of SEQ ID NO:10; c) apolypeptide chosen from SEQ ID NOs:14–72; h) amino acid residue 30 toamino acid residue 63 of SEQ ID NO:2; i) amino acid residue 31 to aminoacid residue 63 of SEQ ID NO:2; j) amino acid residue 30 to amino acidresidue 64 of SEQ ID NO:2; k) amino acid residue 31 to amino acidresidue 64 of SEQ ID NO:2; l) amino acid residue 20 to amino acidresidue 67 of SEQ ID NO:10 and m) amino acid residue 22 to amino acidresidue 67 of SEQ ID NO:10; whereby said polypeptide ameliorates saiddisease. Within one embodiment the microbial-related disease isassociated with the eye. Within a related embodiment themicrobial-related disease is conjunctivitis. Within another embodimentthe microbial-related disease is associated with the ear.

Also provided is a method of contraception comprising administering to amammal a therapeutically effective amount of a polypeptide selected fromthe group consisting of: a) a polypeptide of SEQ ID NO:2; b) apolypeptide of SEQ ID NO:10; c) a polypeptide chosen from SEQ IDNOs:14–72; h) amino acid residue 30 to amino acid residue 63 of SEQ IDNO:2; i) amino acid residue 31 to amino acid residue 63 of SEQ ID NO:2;j) amino acid residue 30 to amino acid residue 64 of SEQ ID NO:2; k)amino acid residue 31 to amino acid residue 64 of SEQ ID NO:2; l) aminoacid residue 20 to amino acid residue 67 of SEQ ID NO:10 and m) aminoacid residue 22 to amino acid residue 67 of SEQ ID NO:10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the three disulfide bond structure of the conservedβ-defensin motif.

FIG. 2 illustrates a multiple alignment of mature, processed human SAP-1(see, for example, Bensch et al., FEBS Lett. 368(2): 331–5, 1995) andthe zamp1 polypeptide of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms.

The term “affinity tag” is used herein to denote a peptide segment thatcan be attached to a polypeptide to provide for purification of thepolypeptide or provide sites for attachment of the polypeptide to asubstrate. In principal, any peptide or protein for which an antibody orother specific binding agent is available can be used as an affinitytag. Affinity tags include a poly-histidine tract, protein A (Nilsson etal., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204–1210,1988; available from Eastman Kodak Co., New Haven, Conn.), streptavidinbinding peptide, or other antigenic epitope or binding domain. See, ingeneral Ford et al., Protein Expression and Purification 2: 95–107,1991. DNAs encoding affinity tags are available from commercialsuppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides and proteins. Where the contextallows, these terms are used with reference to a particular sequence orportion of a polypeptide or protein to denote proximity or relativeposition. For example, a certain sequence positioned carboxyl-terminalto a reference sequence within a protein is located proximal to thecarboxyl terminus of the reference sequence, but is not necessarily atthe carboxyl terminus of the complete protein.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774–78, 1985). When applied to a protein, the term “isolated”indicates that the protein is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated protein is substantially free of other proteins,particularly other proteins of animal origin. It is preferred to providethe protein in a highly purified form, i.e., greater than 95% pure, morepreferably greater than 99% pure.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis or translated from alternatively spliced mRNAs. Receptorpolypeptides are said to be substantially free of transmembrane andintracellular polypeptide segments when they lack sufficient portions ofthese segments to provide membrane anchoring or signal transduction,respectively.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAsequence that encodes a polypeptide having homology to proteins of theβ-defensin family. That is, the zamp1 polypeptides of the presentinvention exhibit a conserved motif shown in SEQ ID NO: 3 and herein:C(X)₆C(X)₄C(X)₇GXC(X)₆CC wherein “(X)” is the number of preferablynon-cysteine amino acid residues between specific amino acids. Thecysteine position and spacing is characteristic of the β-defensinfamily. In addition, the QIG tripeptide motif embedded in the conservedmotif shown in SEQ ID NO:3 occurs in several members of the β-defensinfamily (for example, SAP-1/hBD-2, BNBDS, TAP and the like). This motifis interpreted to indicate the presence of three disulfide bonds in theβ-defensin structure. Those disulfide bonds are shown in FIG. 1. Inaddition, an intron sequence of approximately 900 base pairs is found ingenomic DNA sequence encoding the zamp1 polypeptide. This intronsequence is inserted between the two guanine residues in the codonencoding the glycine residue at amino acid position 20 in SEQ ID NO:10.Such intron placement, in the area between the signal sequence and themature protein occurs in other members of the β-defensin family.

A standard Northern blot tissue distribution of the mRNA correspondingto this novel DNA revealed no expression. It thus appears that normaltissue levels of mRNA of zamp1 polypeptide are below the detectionsensitivity of the Northern blot. Such an observation is consistent withthe knowledge in the art regarding defensins, i.e., that they areconstitutively expressed at low levels but are highly inducible uponinfection. Electronic analysis of tissue distribution based uponlibraries where the sequence is found indicate that zamp1 polypeptide isexpressed in bronchial epithelia.

The novel zamp1 polypeptides of the present invention were initiallyidentified by querying an EST database for homologous sequences to theSAP-1 human defensin isolated from human psoriatic skin. A single ESTsequence was discovered in a bronchial epithelium cDNA library and waspredicted to be related to the β-defensin family. A second search basedupon the β-defensin consensus motif also identified the EST. Thus, theconsensus motif is found in the zamp1 polypeptide as well as in theSAP-1 protein; however, the remaining sequence of the two proteins isdivergent, characterized by approximately 43% identity at the amino acidlevel. See, for example, the multiple alignment shown in FIG. 2.

The nucleotide sequence of the zamp1 polypeptide is described in SEQ IDNO:1 and SEQ ID NO:9, and its deduced amino acid sequence is describedin SEQ ID NO:2 and SEQ ID NO:10, respectively. The zamp1 polypeptide, bysequence analysis, can be grouped with the two human β-defensins, hBD-1and hBD-2 (SAP-1), but it is most closely sequence-related to hBD-2 andthe bovine BNBDs and less similar to hBD-1.

Preliminary computer-aided model building efforts to construct athree-dimensional model structure for zamp1 polypeptide indicate that itis feasible to generate physically reasonable model structures usingBNBD_(—)12 (Zimmermann et al., Biochemistry 34(41): 13663–13671, 1995)as a template. Although there is relatively low sequence identitybetween these two peptides, their overall secondary structure is verysimilar. The most variability is observed in the loop regions, which isnot alarming since the loop segments represent one of many possibleconformations for each loop. Both structures are built primarily of ananti-parallel beta-sheet core, four-stranded in the BNBD_(—)12 model andthree-stranded in the zamp1 polypeptide model. A turn formed between twoof the beta strands in the BNBD_(—)12 chain is also found in the zamp1polypeptide model connecting two beta strands. The overall folding ofBNBD_(—)12 follows the pattern of beta strand/short beta strand/shortbeta strand/turn/beta strand. Folding of zamp1 polypeptide consists ofbeta strand/beta strand/turn/beta strand. These common structuralelements are highly superimposable. Thus, the two polypeptides may wellbe involved in the same or similar biological processes.

Another aspect of the present invention includes zamp1 polypeptidefragments. Preferred fragments include the leader sequence, ranging fromamino acid 1 (Ile) to amino acid 18 (Gly) or 20 (Gly) of SEQ ID NO:2 andranging from amino acid 1 (Met) to amino acid 20 (Gly) or 22 (Gly) ofSEQ ID NO:10. Such leader sequences may be used to direct the secretionof other polypeptides. Such fragments of the present invention may beused as follows: the alternative secretion leader fragments are formedas fusion proteins with alternative proteins selected for secretion;plasmids bearing regulatory regions capable of directing the expressionof the fusion protein are introduced into test cells; and secretion ofthe protein is monitored.

Additional preferred fragments include the β-hairpin loop of the zamp1polypeptide, amino acid 30 (Lys) or 31 (Tyr) to amino acid 63 (Cys) oramino acid 64 (Arg) of SEQ ID NO:2, SEQ ID NO:14, and SEQ ID NOs:18–34.Also provided are the polypeptides of SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NOs:35–72, that eliminate the structural complicationspresented by the free cysteine residues (amino acids 55 and 62 of SEQ IDNO:2) and maintain the hydrophobic face of the polypeptide. Thefragments provided by the invention would be useful in applications,described herein, where administration of zamp1 would be beneficial,such as anti-microbial agents (Thennarasu and Nagaraj, Biochem. Biophys.Res. Comm. 254:281–3, 1999).

The present invention also provides fusion constructs incorporating thezamp1 polypeptide selected from the group consisting of: (a) polypeptidemolecules comprising a sequence of amino acid residues as shown in SEQID NO:2 from amino acid residue 1 (Ile), 19 (His) or 21 (Gly) to aminoacid residue 65 (Lys) or a sequence of amino acid residues as shown inSEQ ID NO: 10 from amino acid residue 1 (Met), 21 (His) or 23 (Gly) toamino acid residue 67 (Lys); or (b) mammalian species homologs or humanparalogs of (a); at least the mature polypeptide region of anotherdefensin molecule; and, optionally, a polypeptide linker there between.When defensin molecules having disparate spectrum of pathogens, fusionconstructs containing the same are expected to exhibit a broader rangeof anti-microbial effectiveness. Polypeptide linkers are preferablyemployed if necessary to provide separation of component polypeptides ofthe fusion or to allow for flexibility of the fusion protein, therebypreserving the anti-microbial activity of each defensin component of thefusion protein. Those of ordinary skill in the art are capable ofdesigning such linkers.

The highly conserved amino acids in the consensus domain of zamp1polypeptide can be used as a tool to identify new family members. Forinstance, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding the conserved motif from RNAobtained from a variety of tissue sources. More specifically, thefollowing probes can be employed to identify other human or zamp1-likeβ-defensins. A preferred embodiment of this aspect of the presentinvention ranges between amino acid residues 31 and 61 of SEQ ID NO: 2(corresponding to nucleotides 91–183 of SEQ ID NO: 1). In particular,highly degenerate primers designed from the above sequences are usefulfor this purpose.

SEQ ID NO: 4 is a degenerate polynucleotide sequence that encompassesall polynucleotides that encode the zamp1 polypeptide of SEQ ID NO: 2(amino acids 1–65). SEQ ID NO: 11 is a degenerate polynucleotidesequence that encompasses all polynucleotides that encode the zamp1polypeptide of SEQ ID NO: 10. Thus, zamp1 polypeptide-encodingpolynucleotides ranging from nucleotide 1, 61 or 67 to nucleotide 195 or213 of SEQ ID NO: 4 and ranging from nucleotide 1, 61 or 67 tonucleotide 201 or 219 of SEQ ID NO: 11 are contemplated by the presentinvention. Also contemplated by the present invention are fragments andfusions as described above with respect to SEQ ID NO: 1 and SEQ ID NO:10, which are formed from analogous regions of SEQ ID NO: 4 and SEQ IDNO: 11. The symbols in SEQ ID NO: 4 are summarized in Table 1 below.

TABLE 1 Nucleotide Resolutions Complement Resolutions A A T T C C G G GG C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G SA|T W A|T W C|G H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|TH A|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO: 4 and SEQ ID NO: 11,encompassing all possible codons for a given amino acid, are set forthin Table 2 below.

TABLE 2 Amino Degenerate Acid Letter Codons Codon Cys C TGC TGT TGY SerS AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCCCCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn NAAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His HCAC CAT CAY Arg R AGA AGG CGA CGC CGG MGN CGT Lys K AAA AAG AAR Met MATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val VGTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGGTer . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap — — —0 —

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO: 2 and SEQ ID NO: 10. Variant sequences can bereadily tested for functionality as described herein.

Within another aspect of the present invention there is provided apharmaceutical composition comprising purified zamp1 polypeptide incombination with a pharmaceutically acceptable vehicle. Suchpharmaceutical compositions are used in the treatment of conditionsassociated with pathological microbes, including bacterial, fungal andviral infections. Antibacterial applications of zamp1 polypeptideinclude situations where the pathogen has become resistant to standardtreatments. For example, hospital sepsis is an increasing problem, sinceStaphylococcus strains have become resistant to commonly usedantibiotics.

In general, anti-microbial activity of zamp1 polypeptides, fragments,fusions, antibodies, agonists and antagonists can be evaluated bytechniques that are known in the art. More specifically, anti-microbialactivity can be assayed by evaluating the sensitivity of microbial cellcultures to test agents and by evaluating the protective effect of testagents on infected mice. See, for example, Musiek et al., Antimicrob.Agents Chemothr. 3: 40, 1973. Antiviral activity can also be assessed byprotection of mammalian cell cultures. Known techniques for evaluatinganti-microbial activity include, for example, Barsum et al., Eur.Respir. J. 8(5): 709–14, 1995; Sandovsky-Losica et al., J. Med. Vet.Mycol (England) 28(4): 279–87, 1990; Mehentee et al., J. Gen. Microbiol(England) 135 (Pt. 8): 2181–8, 1989; Segal and Savage, Journal ofMedical and Veterinary Mycology 24: 477–479, 1986 and the like. Knownassays specific for anti-viral activity include, for example, thosedescribed by Daher et al., J. Virol. 60(3): 1068–74, 1986.

In addition, contract laboratories offer services in evaluatinganti-microbial properties. For example, Panlabs, Inc. of Bothell, Wash.offer in vitro or in vivo testing for bacteria, gram negative(Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella typhimurium and Serratiamarcescens), gram positive (Bacillus subtilis, Brevebacteriumammoniagenes, Corynebacterium minutissimum, Micrococcus luteus,Mycobacterium ranae, Staphylococcus strains and Streptococcus strains)and anaerobic organisms (Actinomyces viscosus, Bacteroides fragilis,Clostridium sporogenes, Corynebacterium acnes, Helicobacter pylori andPorphyromonas gingivalis), as well as for protozoa (Trichomonas foetus)and fungi (e.g., Candida albicans, Epidermophyton floccosum, Exophialajeanselmei, Microsporum strains, Trichophyton strains and the like).Also, Molecular Probes of Oregon has commercially available fluorescencetechnology for use in bacteriology.

If desired, zamp1 polypeptide, fragment, fusion protein, agonist,antagonist or antibody performance in this regard can be compared toproteins known to be functional in this regard, such as proline-richproteins, lysozyme, histatins, lactoperoxidase or the like. In addition,zamp1 polypeptide, fragment, fusion protein, antibody, agonist orantagonist may be evaluated in combination with one or moreanti-microbial agents to identify synergistic effects.

Defensins have been found associated with the tissues and secretions ofthe human eye and are useful in treating microbial-related diseases ofthe eye (Cullor et al., Arch. Ophthalmol. 108:861–4, 1990; Murphy etal., U.S. Pat. No. 5,242,902; Hattenbach et al., Antimicrob. Agent.Chemother. 42:332, 1998; Haynes et al., Lancet 354:451–2, 1998 andHaynes et al., Br. J. Ophthalmol. 83:737–41, 1999). Addition of defensinpolypeptides reduced microbial contamination of corneal preservationmedium. Such agents are useful for reducing infectious postoperativecomplications associated with such transplantations (Schwab et al.,Cornea 11:370–5, 1992).

Thus, zamp1 polypeptides, agonists or antagonists thereof may betherapeutically useful for treatment of infection and inflammationassociated with the eye. To verify the presence of this capability inzamp1 polypeptides, agonists or antagonists of the present invention,such zamp1 polypeptides, agonists or antagonists are evaluated withrespect to their anti-microbial and chemotactic activities according toprocedures known in the art. If desired, zamp1 polypeptide performancein this regard can be compared to other α and β defensins, rabbitneutrophil defensins and the like. In addition, zamp1 polypeptides oragonists or antagonists thereof may be evaluated in combination with oneor more anti-microbial molecules to identify synergistic effects.

Similarly, zamp1 polypeptides of the present invention would also betherapeutically useful for treatment of infection and inflammationassociated with the ear. The invention provides a method of treating amicrobial-related disease comprising administering to a mammal atherapeutically effective amount of a zamp1 polypeptide whereby thepolypeptide ameliorates the disease. Preferably such polypeptidesinclude the β-hairpin loop of the zamp1 polypeptide, amino acid 30 (Lys)or 31 (Tyr) to amino acid 63 (Cys) or amino acid 64 (Arg) of SEQ IDNO:2, SEQ ID NO:14 and SEQ ID NOs:18–34. Additional exemplarypolypeptides include the polypeptides of SEQ ID NO:15, SEQ ID NO:16, SEQID NO:17 and SEQ ID NOs:35–72. One such application would be formicrobial-related disease is associated with the eye, such asconjunctivitis. Application could also be made for diseases associatedwith the ear.

The invention also provides a method of contraception wherein atherapeutically effective amount of the polypeptides of the presentinvention is administered to a mammal. Such administration would beuseful in preventing implantation and/or development of the embryo.

Defensins have also been found to activate the complement cascade(Prohászka et al., Mol Immunol. 34:809–16, 1997 and Prohászka and Füst,Lancet 352:1152, 1998) The complement component C1q plays a role in hostdefense against infectious agents, such as bacteria and viruses. C1q isknown to exhibit several specialized functions. For example, C1qtriggers the complement cascade via interaction with bound antibody orC-reactive protein (CRP). Also, C1q interacts directly with certainbacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid Acomponent of bacterial endotoxin and membranes of certain intracellularorganelles. C1q binding to the C1q receptor is believed to promotephagocytosis. C1q also appears to enhance the antibody formation aspectof the host defense system. See, for example, Johnston, Pediatr. Infect.Dis. J. 12: 933–41, 1993. Thus, complement-activating defensins wouldact as enhanced anti-microbial agents, promoting lysis or phagocytosisof infectious agents.

The anti-microbial activity of defensins is also useful forcontraceptive applications (Sawicki and Mystkovska, Lancet 353:464–5,1999).

The pharmaceutical compositions of the present invention may also beused when pro-inflammatory activity is desired. Applications for suchpro-inflammatory activity include the treatment of chronic tissuedamage, particularly in areas having a limited or damaged vascularsystem, e.g., damage in extremities associated with diabetes. Incontrast, antagonists to zamp1 polypeptides may be useful asanti-inflammatory agents.

Zamp1 polypeptide pharmaceutical compositions of the present inventionmay also be used in the treatment of conditions where stimulation ofimmune responsiveness is desired. Such conditions include the treatmentof patients having incompetent immune systems, such as AIDs patients orindividuals that have undergone chemotherapy, radiation treatment or thelike.

Because zamp1 polypeptide was found in a bronchial epithelia library andcystic fibrosis is characterized by frequent microbial infection,pharmaceutical compositions containing zamp1 polypeptide are alsocontemplated for use in the treatment of lung infections associated withcystic fibrosis. Also contemplated by the present invention areengineered zamp1 polypeptides that are characterized by decreasedsensitivity to salt concentration. Decreased sensitivity to high saltconcentration will preserve anti-microbial activity of engineered zamp1polypeptides in high salt environments, such as in the lung airways ofpatients suffering from cystic fibrosis. In this manner, pharmaceuticalcompositions containing engineered zamp1 polypeptides that areformulated for delivery to the lungs can be used to treat lunginfections associated with cystic fibrosis.

Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245–50, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Commerciallyavailable radiation hybrid mapping panels which cover the entire humangenome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel(Research Genetics, Inc., Huntsville, Ala.), are available. These panelsenable rapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This includes establishing directlyproportional physical distances between newly discovered genes ofinterest and previously mapped markers. The precise knowledge of agene's position can be useful for a number of purposes, including: 1)determining if a sequence is part of an existing contig and obtainingadditional surrounding genetic sequences in various forms, such as YACs,BACs or cDNA clones; 2) providing a possible candidate gene for aninheritable disease which shows linkage to the same chromosomal region;and 3) cross-referencing model organisms, such as mouse, which may aidin determining what function a particular gene might have.

The results showed that the zamp1 gene maps 33.5 cR_(—)3000 from the topof the human chromosome 8 linkage group on the WICGR radiation hybridmap. Proximal and distal framework markers were CHLC.GATA62D10 andWI-3823 (D8S1511), respectively. The use of surrounding markerspositions the zamp1 gene in the 8p23.3–p23.2 region on the integratedLDB chromosome 8 map (located on the Internet, e.g., a public server ofThe Genetic Location Database, University of Southhampton.

Previously, human defensin genes of both hematopoietic (such as HD-1, asdescribed by Sparkes et al., Genomics 5(2): 240–4, 1989) and epithelial(such as HD-5 and HD-6, as described by Bevins et al., Genomics 31(1):95–106, 1996) origin are localized on the short arm of human chromosome8 (8p23). Several defensin genes, cryptidins, have been mapped in themouse genome and are found in a region of conserved synteny with humanon mouse chromosome 8. See, for example, Ouellette et al., Genomics5(2): 233–9, 1989. Recently, Liu et al., Genomics 43(3): 316–20, 1997,reported the mapping of the hBD_(—)1 gene to the same cluster ofdefensins on chromosome 8. These authors propose that α- and β-defensingenes arose from a common ancestral gene prior to mammalian divergence.Thus, the localization of the zamp1 polypeptide-encoding gene to thisregion of chromosome 8 adds a second human β-defensin to the samechromosomal location as the human classic defensins and supports thehypothesis for the evolution of defensins.

The present invention also provides reagents which will find use indiagnostic applications. For example, the zamp1 gene, a probe comprisingzamp1 DNA or RNA or a subsequence thereof can be used to determine ifthe zamp1 gene is present on chromosome 8 or if a mutation has occurred.Detectable chromosomal aberrations at the zamp1 gene locus include butare not limited to aneuploidy, gene copy number changes, insertions,deletions, restriction site changes and rearrangements. Such aberrationscan be detected using polynucleotides of the present invention byemploying molecular genetic techniques, such as restriction fragmentlength polymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel, et. al., ibid.;Marian, Chest, 108: 255–265, 1995).

Another aspect of the present invention involves the detection of zamp1polypeptides in cell culture or in a serum sample or tissue biopsy of apatient undergoing evaluation for SPG, Chediak-Higashi syndrome or otherconditions characterized by an alteration in defensin concentration.Zamp1 polypeptides can be detected using immunoassay techniques andantibodies capable of recognizing a zamp1 polypeptide epitope. Morespecifically, the present invention contemplates methods for detectingzamp1 polypeptide comprising:

exposing a solution or sample or cell culture lysate or supernatant,possibly containing zamp1 polypeptide, to an antibody attached to asolid support, wherein said antibody binds to a first epitope of a zamp1polypeptide;

washing said immobilized antibody-polypeptide to remove unboundcontaminants;

exposing the immobilized antibody-polypeptide to a second antibodydirected to a second epitope of a zamp1 polypeptide, wherein the secondantibody is associated with a detectable label; and

detecting the detectable label. Zamp1 polypeptide concentrationdiffering from that of controls may be indicative of SPG,Chediak-Higashi syndrome or other conditions characterized by analteration in defensin concentration. In addition, expression of zamp1may be monitored in cystic fibrosis patients as a predictor of the onsetof infectious crises. Also, high defensin, such as zamp1 polypeptide,levels have been associated with cytotoxic effects in lung, indicatingthat zamp1 polypeptide levels can be used to direct treatment foraverting or addressing such cytotoxicity. For example, antibodiesdirected to zamp1 polypeptide can be administered to inactivate the samein a treatment modality.

Within additional aspects of the invention there are provided antibodiesor synthesized binding proteins(e.g., those generated by phage display,E. coli Fab, and the like) that specifically bind to the zamp1polypeptides described above. Such antibodies are useful for, amongother uses as described herein, preparation of anti-idiotypicantibodies. Synthesized binding proteins may be produced by phagedisplay using commercially available kits, such as the Ph.D.™ PhageDisplay Peptide Library Kits available from New England Biolabs, Inc.(Beverly, Mass.). Phage display techniques are described, for example,in U.S. Pat. Nos. 5,223,409, 5,403,484 and 5,571,698.

An additional aspect of the present invention provides methods foridentifying agonists or antagonists of the zamp1 polypeptides disclosedabove, which agonists or antagonists may have valuable properties asdiscussed further herein. Within one embodiment, there is provided amethod of identifying zamp1 polypeptide agonists, comprising providingcells responsive thereto, culturing the cells in the presence of a testcompound and comparing the cellular response with the cell cultured inthe presence of the zamp1 polypeptide, and selecting the test compoundsfor which the cellular response is of the same type.

Within another embodiment, there is provided a method of identifyingantagonists of zamp1 polypeptide, comprising providing cells responsiveto a zamp1 polypeptide, culturing a first portion of the cells in thepresence of zamp1 polypeptide, culturing a second portion of the cellsin the presence of the zamp1 polypeptide and a test compound, anddetecting a decrease in a cellular response of the second portion of thecells as compared to the first portion of the cells.

A further aspect of the invention provides a method of studyingchemoattraction of monocytes in cell culture, comprising incubatingmonocytes in a culture medium comprising a zamp1 polypeptide, fragment,fusion protein, antibody, agonist or antagonist to study or evaluatemonocyte chemoattraction. Such evaluation may be conducted using methodsknown in the art, such as those described by Territo et al. referencedabove.

Melanocortin receptors are G-coupled protein receptors which activateadenylate cyclase and cause calcium flux. The agouti protein (whichcontains a 36 amino acid domain that is toxin-like) is thought toinhibit the binding of MSH-alpha to MC1 and MC4. In addition, the agoutiprotein is thought to be an antagonist of calcium channels, and certaintoxins are believed to modulate ion flux. Experimental evidence has beengenerated, suggesting that defensins are capable of blocking calciumchannels.

A further aspect of the invention provides a method of studying activityof the melanocortin family of receptors in cell culture, comprisingincubating cells that endogenously bear such receptors (e.g., ACTHreceptors or the like) or cells that have been engineered to bear suchreceptors in a culture medium comprising a ligand or putative ligand andzamp1 polypeptide, fragment, fusion protein, antibody, agonist orantagonist to study or evaluate ligand or putative ligand binding and/orion flux regulation or modulation. Such evaluation may be conductedusing methods known in the art, such as those described by Zhu et al.referenced above.

A further aspect of the invention provides a method of studying ion fluxin cell culture, comprising incubating cells that are capable of ionflux, such as calcium flux, sodium flux, potassium flux or the like, ina culture medium comprising zamp1 polypeptide, fragment, fusion protein,antibody, agonist or antagonist to study or evaluate ion flux regulationor modulation.

A further aspect of the invention provides a method of studyingcytocidal activity against mammalian cells, such as tumor cells, in cellculture, comprising incubating such cells in a culture medium comprisinga zamp1 polypeptide, fragment, fusion protein, antibody, agonist orantagonist at high test agent and low cell concentration to study orevaluate cytocidal activity. Such evaluation may be conducted usingmethods known in the art, such as those described by Lichtenstein etal., Blood 68: 1407–10, 1986 and Sheu et al., Antimicrob. AgentsChemother. 28: 626–9, 1993.

Another aspect of the present invention involves the use of zamp1polypeptides, fragments, fusion proteins or agonists as cell culturereagents in in vitro studies of exogenous microorganism infection, suchas bacterial, viral or fungal infection. Such moieties may also be usedin in vivo animal models of infection.

An additional aspect of the present invention is to study epithelialcell defensin induction in cell culture. In this aspect of the presentinvention, epithelial cells are cultured and exposed to pathogenicstimuli. Induction of zamp1 polypeptide production by the epithelialcells is then measured.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO: 10, other probe sequences specifically set forthherein, or a sequence complementary thereto, under stringent conditions.In general, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Typical stringent conditionsare those in which the salt concentration is up to about 0.03 M at pH 7and the temperature is at least about 60° C.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for isolating DNA and RNA arewell known in the art. It is generally preferred to isolate RNA frombronchial epithelium, although DNA can also be prepared using RNA fromother tissues or isolated as genomic DNA. Total RNA can be preparedusing guanidine HCl extraction followed by isolation by centrifugationin a CsCl gradient (Chirgwin et al., Biochemistry 18:52–94, 1979). Poly(A)⁺ RNA is prepared from total RNA using the method of Aviv and Leder(Proc. Natl. Acad. Sci. USA 69:1408–1412, 1972). Complementary DNA(cDNA) is prepared from poly(A)⁺ RNA using known methods.Polynucleotides encoding zamp1 polypeptides are then identified andisolated by, for example, hybridization or PCR.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zamp1 polypeptides from other mammalian species, includingmurine, rat, porcine, ovine, bovine, canine, feline, equine and otherprimate proteins. Species homologs of the human proteins can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses the protein. Suitable sources of mRNA can be identified byprobing Northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue ofcell line. A zamp1 polypeptide-encoding cDNA can then be isolated by avariety of methods, such as by probing with a complete or: partial humancDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the sequences disclosed herein. Within an additionalmethod, the cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to zamp1 polypeptide. Similar techniques can also be applied tothe isolation of genomic clones.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:9 and SEQ ID NO:10 represent asingle allele of the human zamp1 gene and polypeptide, and that allelicvariation and alternative splicing are expected to occur. Allelicvariants can be cloned by probing cDNA or genomic libraries fromdifferent individuals according to standard procedures. Allelic variantsof the DNA sequence shown in SEQ ID NO:2 and SEQ ID NO:10, includingthose containing silent mutations and those in which mutations result inamino acid sequence changes, are within the scope of the presentinvention.

Within preferred embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules having at least a portion of the nucleotide sequence of SEQ IDNOs:1, 4, 9 or 11 or to nucleic acid molecules having a nucleotidesequence complementary to those sequences. In general, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH. The T_(m) is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1–1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5–25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20–70° C. and a hybridization buffer containingup to 6×SSC and 0–50% formamide. A higher degree of stringency can beachieved at temperatures of from 40–70° C. with a hybridization bufferhaving up to 4×SSC and from 0–50% formamide. Highly stringent conditionstypically encompass temperatures of 42–70° C. with a hybridizationbuffer having up to 1×SSC and 0–50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions which influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software, such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20–25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5–10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration ofthe buffer, the stability of the hybrid is increased. Typically,hybridization buffers contain from between 10 mM–1 M Na⁺. The additionof destabilizing or denaturing agents such as formamide,tetralkylammonium salts, guanidinium cations or thiocyanate cations tothe hybridization solution will alter the T_(m) of a hybrid. Typically,formamide is used at a concentration of up to 50% to allow incubationsto be carried out at more convenient and lower temperatures. Formamidealso acts to reduce non-specific background when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant zamp1polypeptide can be hybridized with a nucleic acid molecule having atleast a portion of the nucleotide sequence of SEQ ID NOs:1, 4, 9 or 11(or their complements) at 42° C. overnight in a solution comprising 50%formamide, 5×SSC (1×SSC: 0.15 M sodium chloride and 15 mM sodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution (100×Denhardt's solution: 2% (w/v) FICOLL® 400, 2% (w/v)polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA. One of skillin the art can devise variations of these hybridization conditions. Forexample, the hybridization mixture can be incubated at a higher or lowertemperature, such as about 65° C., in a solution that does not containformamide. Moreover, premixed hybridization solutions are available(e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories,Inc.), and hybridization can be performed according to themanufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×–2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55–65° C. That is, nucleic acid moleculesencoding a variant zamp1 polypeptide hybridize with a nucleic acidmolecule having at least a portion of the nucleotide sequence of SEQ IDNOs:1, 4, 9 or 11 (or their complements) under stringent washingconditions, in which the wash stringency is equivalent to 0.5×–2×SSCwith 0.1% SDS at 50–65° C., including 0.5×SSC with 0.1% SDS at 55° C.,or 2×SSC with 0.1% SDS at 65° C. One of skill in the art can readilydevise equivalent conditions, for example, by substituting SSPE for SSCin the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×–0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50–65° C. In other words, nucleic acid molecules encoding a variantzamp1 polypeptide hybridize with a nucleic acid molecule having at leasta portion of the nucleotide sequence of SEQ ID NOs:1, 4, 9 or 11 (ortheir complements) under highly stringent washing conditions, in whichthe wash stringency is equivalent to 0.1×–0.2×SSC with 0.1% SDS at50–65° C., including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with0.1% SDS at 65° C.

The present invention also contemplates zamp1 variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NOs:2 or 10, and a hybridization assay, as describedabove. Such zamp1 variants include nucleic acid molecules (1) thathybridize with at least a portion of a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1, 4, 9 or 11 (or their complements)under stringent washing conditions, in which the wash stringency isequivalent to 0.5×–2×SSC with 0.1% SDS at 50–65° C., and (2) that encodea polypeptide having at least 80%, at least 90%, at least 95% or greaterthan 95% sequence identity to the amino acid sequence of SEQ ID NOs:2 or10. Alternatively, zamp1 variants can be characterized as nucleic acidmolecules (1) that hybridize with a nucleic acid molecule having atleast a portion of the nucleotide sequence of SEQ ID NO:1, 4, 9 or 11(or their complements) under highly stringent washing conditions, inwhich the wash stringency is equivalent to 0.1×–0.2×SSC with 0.1% SDS at50–65° C., and (2) that encode a polypeptide having at least 80%, atleast 90%, at least 95% or greater than 95% sequence identity to theamino acid sequence of SEQ ID NOs:2 or 10.

The present invention also provides isolated zamp1 polypeptides that aresubstantially homologous to the polypeptides of SEQ ID NO:2 and SEQ IDNO:10 and their species homologs/orthologs. The term “substantiallyhomologous” is used herein to denote polypeptides having 50%, preferably60%, more preferably at least 80%, sequence identity to the sequencesshown in SEQ ID NO:2 or SEQ ID NO:10 or their orthologs or paralogs.Such polypeptides will more preferably be at least 90% identical, andmost preferably 95% or more identical to SEQ ID NO:2 or SEQ ID NO:10 orits orthologs or paralogs. Percent sequence identity is determined byconventional methods. See, for example, Altschul et al., Bull. Math.Bio. 48: 603–616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915–10919, 1992. Briefly, two amino acid sequences are alignedto optimize the alignment scores using a gap opening penalty of 10, agap extension penalty of 1, and the “blosum 62” scoring matrix ofHenikoff and Henikoff (ibid.) as shown in Table 3 (amino acids areindicated by the standard one-letter codes). The percent identity isthen calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{matrix}\begin{matrix}\left\lbrack {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \right. \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}}\end{matrix} \\\left. {{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zamp1. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat. Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol.183:63, 1990.

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NOs:2 or 10) and atest sequence that have either the highest density of identities (if thektup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen re-scored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63, 1990.

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

The present invention includes nucleic acid molecules that encode apolypeptide having one or more “conservative amino acid substitutions,”compared with the amino acid sequence of SEQ ID NOs:2 or 10.Conservative amino acid substitutions can be based upon the chemicalproperties of the amino acids. That is, variants can be obtained thatcontain one or more amino acid substitutions of SEQ ID NOs:2 or 10, inwhich an alkyl amino acid is substituted for an alkyl amino acid in azamp1 amino acid sequence, an aromatic amino acid is substituted for anaromatic amino acid in a zamp1 amino acid sequence, a sulfur-containingamino acid is substituted for a sulfur-containing amino acid in a zamp1amino acid sequence, a hydroxy-containing amino acid is substituted fora hydroxy-containing amino acid in a zamp1 amino acid sequence, anacidic amino acid is substituted for an acidic amino acid in a zamp1amino acid sequence, a basic amino acid is substituted for a basic aminoacid in a zamp1 amino acid sequence, or a dibasic monocarboxylic aminoacid is substituted for a dibasic monocarboxylic amino acid in a zamp1amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. Table 4 also provided commonconservative amino acid substitutions.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1992). Accordingly, the BLOSUM62 substitution frequencies can be used todefine conservative amino acid substitutions that may be introduced intothe amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Conservative amino acid changes in a zamp1 gene can be introduced bysubstituting nucleotides for the nucleotides recited in SEQ ID NOs:1 or9. Such “conservative amino acid” variants can be obtained, for example,by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995) at pages 8–10 to 8–22; and McPherson (ed.), DirectedMutagenesis: A Practical Approach (IRL Press 1991)). The ability of suchvariants to promote the anti-microbial or other properties of thewild-type protein can be determined using a standard methods, such asthe assays described herein. Alternatively, a variant zamp1 polypeptidecan be identified by the ability to specifically bind anti-zamp1antibodies.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 4) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20–25 residues, or a small extension that facilitates purification (anaffinity tag), such as a poly-histidine tract, protein A (Nilsson etal., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988),maltose binding protein (Kellerman and Ferenci, Methods Enzymol.90:459–463, 1982; Guan et al., Gene 67:21–30, 1987), thioredoxin,ubiquitin, cellulose binding protein, T7 polymerase, or other antigenicepitope or binding domain. See, in general Ford et al., ProteinExpression and Purification 2: 95–107, 1991. DNAs encoding affinity tagsare available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.; New England Biolabs, Beverly, Mass.). Polypeptidescomprising affinity tags can further comprise a proteolytic cleavagesite between the zamp1 polypeptide and the affinity tag. Preferred suchsites include thrombin cleavage sites and factor Xa cleavage sites.

TABLE 4 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The proteins of the present invention can also comprise, in addition tothe 20 standard amino acids, non-naturally occurring amino acidresidues. Non-naturally occurring amino acids include, withoutlimitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl-glycine,allo-threonine, methylthreonine, hydroxyethyl-cysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenyl-alanine, 4-fluorophenylalanine, 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and •-methyl serine. Severalmethods are known in the art for incorporating non-naturally occurringamino acid residues into proteins. For example, an in vitro system canbe employed wherein nonsense mutations are suppressed using chemicallyaminoacylated suppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations are carried out in a cell freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Meth. Enzymol. 202:301, 1991; Chung et al., Science259:806–09, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145–49, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991–98, 1996). Within a third method, E. coli cells are culturedin the absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470–76, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395–403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zamp1 polypeptide aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, or preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in the zamp1 polypeptides of the present inventioncan be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081–1085, 1989). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity (e.g., anti-microbial activity) to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., J. Biol. Chem. 271:4699–4708, 1996. Sites of ligand-receptor orother biological interaction can also be determined by physical analysisof structure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., Science 255:306–312, 1992; Smithet al., J. Mol. Biol. 224:899–904, 1992; Wlodaver et al., FEBS Lett.309:59–64, 1992. The identities of essential amino acids can also beinferred from analysis of homologies with related β-defensins.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53–57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152–2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832–10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zamp1 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389–91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747–51, 1994and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated byin vitro homologous recombination by random fragmentation of a parentDNA followed by reassembly using PCR, resulting in randomly introducedpoint mutations. This technique can be modified by using a family ofparent DNAs, such as allelic variants or DNAs from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides (e.g., anti-microbialactivity) can be recovered from the host cells and rapidly sequencedusing modern equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are substantiallyhomologous to residues 1 to 65 of SEQ ID NO: 2 or to residues 1 to 67 ofSEQ ID NO: 10, residues 30 or 31 to residues 63 or 64 of SEQ ID NO:2 orpolypeptides of SEQ ID NOs:14, 15 or 16, or allelic variants thereof andretain the anti-microbial properties of the wild-type protein. Suchpolypeptides may include additional amino acids from affinity tags andthe like. Such polypeptides may also include additional polypeptidesegments as generally disclosed above.

The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.However, host cells must be selected with some care as a result of theanti-microbial activity of the molecules of the present invention. Forexample, any cell culture-based system must be evaluated, because zamp1polypeptides, fragments, fusion proteins, antibodies, agonists orantagonists may kill the host cell as a part of an anti-microbialfunction. Zamp1 polypeptides are of a small enough size to permitpreparation by PCR or other protein chemistry techniques to avoid anypotential host cell toxicity problems. Alternatively, native orengineered precursor proteins, prior to post-translational cleavage toyield the mature zamp1 polypeptide, are inactive, thereby limiting hostcell cytotoxicity prior to lysosomal packaging. See, for example, Lehreret al., Cell 64: 229–30, 1991. Thus, precursor proteins to zamp1polypeptides may be produced in microbial cell culture.

Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryoticcells, particularly cultured cells of multicellular organisms, arepreferred. Techniques for manipulating cloned DNA molecules andintroducing exogenous DNA into a variety of host cells are disclosed bySambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, andAusubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zamp1 polypeptide of the presentinvention is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a zamp1 polypeptide into the secretory pathway of a host cell,a secretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) is provided in the expression vector. Thesecretory signal sequence may be that of the zamp1 polypeptide, or maybe derived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the zamp1polypeptide-encoding DNA sequence in the correct reading frame.Secretory signal sequences are commonly positioned 5′ to the DNAsequence encoding the polypeptide of interest, although certain signalsequences may be positioned elsewhere in the DNA sequence of interest(see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S.Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid residues1–18 or 12 of SEQ ID NO:2 or amino acid residues 1–20 or 22 of SEQ IDNO:10, is operably linked to another polypeptide using methods known inthe art and disclosed herein. The secretory signal sequence contained inthe fusion polypeptides of the present invention is preferably fusedamino-terminally to an additional peptide to direct the additionalpeptide into the secretory pathway. Such constructs have numerousapplications known in the art. For example, these novel secretory signalsequence fusion constructs can direct the secretion of an activecomponent of a normally non-secreted protein, such as a receptor. Suchfusions may be used in vivo or in vitro to direct peptides through thesecretory pathway.

Cultured mammalian cells are also preferred hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841–845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., eds., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY, 1987), liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al.;Focus 15:80, 1993), and viral vectors (A. Miller and G. Rosman,BioTechniques 7:980–90, 1989; Q. Wang and M. Finer, Nature Med.2:714–16, 1996). The production of recombinant polypeptides in culturedmammalian cells is disclosed, for example, by Levinson et al., U.S. Pat.No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Preferredcultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59–72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47–58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). DNA encoding the zamp1 polypeptide is inserted into thebaculoviral genome in place of the AcNPV polyhedrin gene coding sequenceby one of two methods. The first is the traditional method of homologousDNA recombination between wild-type AcNPV and a transfer vectorcontaining the zamp1 flanked by AcNPV sequences. Suitable insect cells,e.g. SF9 cells, are infected with wild-type AcNPV and transfected with atransfer vector comprising a zamp1 polynucleotide operably linked to anAcNPV polyhedrin gene promoter, terminator, and flanking sequences. See,King and Possee, The Baculovirus Expression System: A Laboratory Guide,London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors:A Laboratory Manual, New York, Oxford University Press., 1994; and,Richardson, C. D., Ed., Baculovirus Expression Protocols. Methods inMolecular Biology, Totowa, N.J., Humana Press, 1995. Naturalrecombination within an insect cell will result in a recombinantbaculovirus which contains zamp1 driven by the polyhedrin promoter.Recombinant viral stocks are made by methods commonly used in the art.

The second method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow et al., J Virol.67:4566–79, 1993). This system is sold in the Bac-to-Bac kit (LifeTechnologies, Rockville, Md.). This system utilizes a transfer vector,pFastBac1™ (Life Technologies) containing a Tn7 transposon to move theDNA encoding the zamp1 polypeptide into a baculovirus genome maintainedin E. coli as a large plasmid called a “bacmid.” The pFastBac1™ transfervector utilizes the AcNPV polyhedrin promoter to drive the expression ofthe gene of interest, in this case zamp1. However, pFastBac1™ can bemodified to a considerable degree. The polyhedrin promoter can beremoved and substituted with the baculovirus basic protein promoter(also known as Pcor, p6.9 or MP promoter) which is expressed earlier inthe baculovirus infection, and has been shown to be advantageous forexpressing secreted proteins. See, Hill-Perkin and Possee, J. Gen.Virol. 71:971–6, 1990; Bonning. et al., J. Gen. Virol. 75:1551–6, 1994;and, Chazenbalk and Rapoport, J. Biol. Chem. 270:1543–9, 1995. In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native zamp1 secretory signal sequences with secretorysignal sequences derived from insect proteins. For example, a secretorysignal sequence from Ecdysteroid Glucosyltransferase (EGT), honey beeMelittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67(PharMingen, San Diego, Calif.) can be used in constructs to replace thenative zamp1 secretory signal sequence. In addition, transfer vectorscan include an in-frame fusion with DNA encoding an epitope tag at theC- or N-terminus of the expressed zamp1 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer et al., Proc. Natl. Acad. Sci.82:7952–4, 1985). Using a technique known in the art, a transfer vectorcontaining zamp1 is transformed into E. coli, and screened for bacmidswhich contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus thatexpresses zamp1 is subsequently produced. Recombinant viral stocks aremade by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells. Thecells are grown up from an inoculation density of approximately 2–5×10⁵cells to a density of 1–2×10⁶ cells at which time a recombinant viralstock is added at a multiplicity of infection (MOI) of 0.1 to 10, moretypically near 3. The recombinant virus-infected cells typically producethe recombinant zamp1 polypeptide at 12–72 hours post-infection andsecrete it with varying efficiency into the medium. The culture isusually harvested 48 hours post-infection. Centrifugation is used toseparate the cells from the medium (supernatant). The supernatantcontaining the zamp1 polypeptide is filtered through micropore filters,usually 0.45 μm pore size. Procedures used are generally described inavailable laboratory manuals (King and Possee, ibid.; O'Reilly et al.,ibid.; Richardson, ibid.). Subsequent purification of the zamp1polypeptide from the supernatant can be achieved using methods describedherein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459–65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinantproteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, it is preferred that thepromoter and terminator in the plasmid be that of a P. methanolica gene,such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Otheruseful promoters include those of the dihydroxyacetone synthase (DHAS),formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitateintegration of the DNA into the host chromosome, it is preferred to havethe entire expression segment of the plasmid flanked at both ends byhost DNA sequences. A preferred selectable marker for use in Pichiamethanolica is a P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), whichallows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is preferred to use host cells in which bothmethanol utilization genes (AUG1 and AUG2) are deleted. For productionof secreted proteins, host cells deficient in vacuolar protease genes(PEP4 and PRB1) are preferred. Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. It is preferred to transform P.methanolica cells by electroporation using an exponentially decaying,pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (τ) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zamp1polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the latter case, the polypeptidecan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) to release the contents of the periplasmic space and recoveringthe protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine)

Expressed recombinant zamp1 polypeptides (or chimeric zamp1polypeptides) can be purified using fractionation and/or conventionalpurification methods and media. Ammonium sulfate precipitation and acidor chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitableanion exchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow SEPHAROSE® (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-SEPHAROSE® FF (Pharmacia),TOYOPEARL® butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-SEPHAROSE®(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their structural properties. For example, immobilizedmetal ion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins or proteins having a His-affinity tag. Briefly,a gel is first charged with divalent metal ions to form a chelate(Sulkowski, Trends in Biochem. 3:1–7, 1985). Histidine-rich proteinswill be adsorbed to this matrix with differing affinities, dependingupon the metal ion used, and will be eluted by competitive elution,lowering the pH, or use of strong chelating agents. Other methods ofpurification include purification of glycosylated proteins by lectinaffinity chromatography and ion exchange chromatography (Methods inEnzymol., Vol. 182, “Guide to Protein Purification”, M. Deutscher,(ed.), Acad. Press, San Diego, 1990, pp.529–39). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

Protein refolding (and optionally reoxidation) procedures may beadvantageously used. It is preferred to purify the protein to >80%purity, more preferably to >90% purity, even more preferably >95%, andparticularly preferred is a pharmaceutically pure state, that is greaterthan 99.9% pure with respect to contaminating macromolecules,particularly other proteins and nucleic acids, and free of infectiousand pyrogenic agents. Preferably, a purified protein is substantiallyfree of other proteins, particularly other proteins of animal origin.

Zamp1 polypeptides or fragments thereof may also be prepared throughchemical synthesis. Zamp1 polypeptides may be monomers or multimers;glycosylated or non-glycosylated; pegylated or non-pegylated; amidatedor non-amidated; sulfated or non-sulfated; and may or may not include aninitial methionine amino acid residue. For example, zamp1 polypeptidescan be synthesized by exclusive solid phase synthesis, partial solidphase methods, fragment condensation or classical solution synthesis.The polypeptides are preferably prepared by solid phase peptidesynthesis, for example as described by Merrifield, J. Am. Chem. Soc.85:2149, 1963. The synthesis is carried out with amino acids that areprotected at the alpha-amino terminus. Trifunctional amino acids withlabile side-chains are also protected with suitable groups to preventundesired chemical reactions from occurring during the assembly of thepolypeptides. The alpha-amino protecting group is selectively removed toallow subsequent reaction to take place at the amino-terminus. Theconditions for the removal of the alpha-amino protecting group do notremove the side-chain protecting groups.

The alpha-amino protecting groups are those known to be useful in theart of stepwise polypeptide synthesis. Included are acyl type protectinggroups (e.g., formyl, trifluoroacetyl, acetyl), aryl type protectinggroups (e.g., biotinyl), aromatic urethane type protecting groups [e.g.,benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxycarbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexloxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tBoc and Fmoc.

The side-chain protecting groups selected must remain intact duringcoupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

In tBoc chemistry, the preferred side-chain protecting groups are tosylfor arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-Cl-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-penta-methyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutamine and histidine, tert-butyl foraspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc forlysine and tryptophan.

For the synthesis of phosphopeptides, either direct or post-assemblyincorporation of the phosphate group is used. In the directincorporation strategy, the phosphate group on serine, threonine ortyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphoramidite and then oxidized bytert-butylhydroperoxide.

Solid phase synthesis is usually carried out from the carboxyl-terminusby coupling the alpha-amino protected (side-chain protected) amino acidto a suitable solid support. An ester linkage is formed when theattachment is made to a chloromethyl, chlorotrityl or hydroxymethylresin, and the resulting polypeptide will have a free carboxyl group atthe C-terminus. Alternatively, when an amide resin such asbenzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry)and Rink amide or PAL resin (for Fmoc chemistry) are used, an amide bondis formed and the resulting polypeptide will have a carboxamide group atthe C-terminus. These resins, whether polystyrene- or polyamide-based orpolyethyleneglycol-grafted, with or without a handle or linker, with orwithout the first amino acid attached, are commercially available, andtheir preparations have been described by Stewart et al., “Solid PhasePeptide Synthesis” (2nd Edition), (Pierce Chemical Co., Rockford, Ill.,1984) and Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton etal., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,Oxford, 1989.

The C-terminal amino acid, protected at the side chain if necessary, andat the alpha-amino group, is attached to a hydroxylmethyl resin usingvarious activating agents including dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). Itcan be attached to chloromethyl or chlorotrityl resin directly in itscesium tetramethylammonium salt form or in the presence of triethylamine(TEA) or diisopropylethylamine (DIEA). First amino acid attachment to anamide resin is the same as amide bond formation during couplingreactions.

Following the attachment to the resin support, the alpha-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300–320 nm or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH2Cl2 or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970.

After the entire assembly of the desired peptide, the peptide-resin iscleaved with a reagent with proper scavengers. The Fmoc peptides areusually cleaved and deprotected by TFA with scavengers (e.g., H2O,ethanedithiol, phenol and thioanisole). The tBoc peptides are usuallycleaved and deprotected with liquid HF for 1–2 hours at −5 to 0° C.,which cleaves the polypeptide from the resin and removes most of theside-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury(II)acetate and alternatively by iodine, thallium(III)trifluoroacetate or silver tetrafluoroborate which simultaneouslyoxidize cysteine to cystine. Other strong acids used for tBoc peptidecleavage and deprotection include trifluoromethanesulfonic acid (TFMSA)and trimethylsilyltrifluoroacetate (TMSOTf).

A zamp1 polypeptide ligand-binding polypeptide can also be used forpurification of ligand. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligandto bind to the receptor polypeptide. The ligand is then eluted usingchanges in salt concentration, chaotropic agents (guanidine HCl), or pHto disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument (BIAcore™,Pharmacia Biosensor, Piscataway, N.J.) may be advantageously employed.Such receptor, antibody, member of a complement/anti-complement pair orfragment is immobilized onto the surface of a receptor chip. Use of thisinstrument is disclosed by Karlsson, J. Immunol. Methods 145:229–40,1991 and Cunningham and Wells, J. Mol. Biol. 234:554–63, 1993. Areceptor, antibody, member or fragment is covalently attached, usingamine or sulfhydryl chemistry, to dextran fibers that are attached togold film within the flow cell. A test sample is passed through thecell. If a ligand, epitope, or opposite member of thecomplement/anti-complement pair is present in the sample, it will bindto the immobilized receptor, antibody or member, respectively, causing achange in the refractive index of the medium, which is detected as achange in surface plasmon resonance of the gold film. This system allowsthe determination of on- and off-rates, from which binding affinity canbe calculated, and assessment of stoichiometry of binding.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660–72, 1949) and calorimetric assays (Cunningham et al., Science253:545–48, 1991; Cunningham et al., Science 245:821–25, 1991).

Zamp1 polypeptides can also be used to prepare antibodies thatspecifically bind to zamp1 polypeptide epitopes, peptides orpolypeptides. Methods for preparing polyclonal and monoclonal antibodiesare well known in the art (see, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982).As would be evident to one of ordinary skill in the art,polyclonal-antibodies can be generated from a variety of warm-bloodedanimals, such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats.

The immunogenicity of a zamp1 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of zamp1 polypeptideor a portion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingonly non-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Alternative techniques forgenerating or selecting antibodies useful herein include in vitroexposure of lymphocytes to zamp1 protein or peptide, and selection ofantibody display libraries in phage or similar vectors (for instance,through use of immobilized or labeled zamp1 protein or peptide).

Antibodies are defined to be specifically binding if: 1) they exhibit athreshold level of binding activity, and/or 2) they do not significantlycross-react with related polypeptide molecules. First, antibodies hereinspecifically bind if they bind to a zamp1 polypeptide, peptide orepitope with a binding affinity (Ka) of 10⁶ mol⁻¹ or greater, preferably10⁷ mol⁻¹ or greater, more preferably 10⁸ mol⁻¹ or greater, and mostpreferably 10⁹ mol⁻¹ or greater. The binding affinity of an antibody canbe readily determined by one of ordinary skill in the art, for example,by Scatchard analysis (G. Scatchard, Ann. NY Acad. Sci. 51: 660–72,1949).

Second, antibodies specifically bind if they do not significantlycross-react with related polypeptides. Antibodies do not significantlycross-react with related polypeptide molecules, for example, if theydetect human zamp1 polypeptide, but not known related polypeptides usinga standard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are orthologs, that is, proteins from thesame species that are members of a protein family, such as other knownhuman β-defensins (e.g., hBD-1 and hBD-2), mutant human β-defensins, andnon-human β-defensins.

Moreover, antibodies may be “screened against” known relatedpolypeptides to isolate a population that specifically binds to theinventive polypeptides. For example, antibodies raised to human zamp1polypeptide are adsorbed with related polypeptides adhered to aninsoluble matrix; antibodies specific to human zamp1 polypeptide willflow through the matrix under the proper buffer conditions. Suchscreening allows isolation of polyclonal and monoclonal antibodiesnon-crossreactive to closely related polypeptides (see, Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988; Current Protocols in Immunology, Cooligan et al. (eds.),National Institutes of Health, John Wiley and Sons, Inc., 1995).Screening and isolation of specific antibodies is well known in the art(see, Fundamental Immunology, Paul (ed.), Raven Press, 1993; Getzoff etal., Adv. Immunol. 43:1–98, 1988; Monoclonal Antibodies: Principles andPractice, Goding, J. W. (eds.), Academic Press Ltd., 1996; Benjamin etal., Ann. Rev. Immunol. 2:67–101, 1984).

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zamp1 proteins or peptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press,1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zamp1protein or peptide.

Antibodies to zamp1 polypeptides may be used for tagging cells thatexpress zamp1 polypeptides; for isolating zamp1 polypeptides by affinitypurification; for diagnostic assays for determining circulating levelsof zamp1 polypeptides; for detecting or quantitating soluble zamp1polypeptide as a marker of underlying pathology or disease; inanalytical methods employing FACS; for screening expression libraries;for generating anti-idiotypic antibodies; and as neutralizing antibodiesor as antagonists to block anti-microbial activity in vitro and in vivo.

For pharmaceutical use, the proteins of the present invention areformulated for topical, inhalant or parenteral, particularly intravenousor subcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a zamp1 protein in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 19th ed., 1995. Therapeutic doses will generally determinedby the clinician according to accepted standards, taking into accountthe nature and severity of the condition to be treated, patient traits,etc. Determination of dose is within the level of ordinary skill in theart. The proteins may be administered for acute treatment, over one weekor less, often over a period of one to three days or may be used inchronic treatment, over several months or years.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Extension of EST Sequence

The novel zamp1 polypeptides of the present invention were initiallyidentified by querying an EST database for homologous sequences to theSAP-1 human defensin isolated from human psoriatic skin. A single ESTsequence was discovered in a bronchial epithelium cDNA library and waspredicted to be related to the β-defensin family. A second search basedupon the β-defensin consensus motif also identified the EST.

To identify the corresponding cDNA, a clone containing the EST wassought, but was not located. Oligonucleotides ZC14741 (SEQ ID NO: 5),ZC14740 (SEQ ID NO: 6) were used in a PCR reaction to isolate the zamp1polypeptide-encoding sequence from human genomic DNA. Reactionconditions were 94° C. for 1 minute and 30 seconds, followed by 35cycles of 94° C. for 10 seconds, 58° C. for 20 seconds and 72° C. for 20seconds, followed by 72° C. for ten minutes. As a template, 100 ng ofhuman genomic DNA was used, and Clontech Advantage PCR mix (Clontech,Palo Alto, Calif.) was used as the polymerase mixture. The resulting 113bp fragment was then purified on a 3.2% NuSieve (FMC Bioproducts,Rockland, Me.) gel using a QiaexII Gel Extraction Kit (Qiagen, Inc.,Chatsworth, Calif.) according to the manufacturer's directions. Thepurified material was used as a template for sequencing. The templatewas sequenced on an ABIPRISM™ model 377 DNA sequencer (Perkin-ElmerCetus, Norwalk, Conn.) using the ABI PRISM™ Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer Corp.) according tomanufacturer's instructions. Oligonucleotides ZC14741 (SEQ ID NO: 5),ZC14740 (SEQ ID NO: 6) were used as primers for sequencing the clone.Sequencing reactions were carried out in a Hybaid OmniGene TemperatureCycling System (National Labnet Co., Woodbridge, N.Y.). SEQUENCHER™ 3. 1sequence analysis software (Gene Codes Corporation, Ann Arbor, Mich.)was used for data analysis. The resulting 113 bp sequence is disclosedin SEQ ID NO: 1. Comparison of the originally derived EST sequence withthe sequence represented in SEQ ID NO: 1 showed that there were 2 basepair differences which resulted in 1 amino acid difference between thededuced amino acid sequences. Note that one of the base pair differenceswere from unknown “N” residues in the EST sequence to known residues inSEQ ID NO: 1.

Generally, one or a combination of several techniques could be used toobtain the full length sequence of the zamp1 polypeptide-encodingpolynucleotide. First, if one or more additional ESTs are identifiedthat contig to the clone sequenced above, clones corresponding to suchESTs can be ordered and sequenced as described above and splicedtogether with the original sequence to form the full length sequence. Ifa small portion of the full length sequence is absent, 5′ RACE reactionscan be done, and the resulting fragments can be sequenced as describedabove and spliced together with the original sequence to form the fulllength sequence. Also, one or more cDNA libraries can be probed with allor a portion of SEQ ID NO: 1 to identify a putative full-length clone.Such a full length clone can be sequenced as described above.

EXAMPLE 2 Tissue Distribution

Northerns were performed using Human Multiple Tissue Blots from Clontech(Palo Alto, Calif.). An approximately 113 bp DNA probe, based directlyon the identified EST, was generated using PCR techniques, specificallya 35 cycle reaction with an annealing temperature of 58° C. usingClontech Advantage KlenTaq Polymerase mix (Clontech). The DNA probe wasradioactively labeled with ³²P using REDIPRIME™ DNA labeling system(Amersham, Arlington Heights, Ill.) according to the manufacturer'sspecifications. The probe was purified using a NUCTRAP push column(Stratagene Cloning Systems, La Jolla, Calif.). EXPRESSHYB (Clontech)solution was used for prehybridization and as a hybridizing solution forthe Northern blots. Hybridization took place overnight at 55° C., andthe blots were then washed in 2×SSC and 0.1% SDS at RT, followed by awash in 0.1×SSC and 0.1% SDS at 50° C. No expression was observed. Itthus appears that normal tissue levels of mRNA of zamp1 polypeptide arebelow the detection sensitivity of the Northern blot. Such anobservation is consistent with the knowledge in the art regarding somedefensins, i.e., that they are constitutively expressed at low levelsbut are highly inducible upon infection.

EXAMPLE 3 Chromosomal Mapping of the Zamp1 Gene

The zamp1 gene was mapped to chromosome 8 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panelcontains PCRable DNAs from each of 93 radiation hybrid clones, plus twocontrol DNAs (the HFL donor and the A23 recipient). A publicly availableWWW server located on the Internet allows mapping relative to theWhitehead Institute/MIT Center for Genome Research's radiation hybridmap of the human genome (the “WICGR” radiation hybrid map) which wasconstructed with the GeneBridge 4 Radiation Hybrid Panel.

For the mapping of the zamp1 gene with the “GeneBridge 4 RH Panel”, 20μl reactions were set up in a PCRable 96-well microtiter plate(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC 14,780 (SEQ ID NO: 7), 1 μl antisenseprimer, ZC 14,776 (SEQ ID NO: 8), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and x μl ddH2O for a total volume of 20 μl. Thereactions were overlaid with an equal amount of mineral oil and sealed.The PCR cycler conditions were as follows: an initial 1 cycle 5 minutedenaturation at 95° C., 35 cycles of a 1 minute denaturation at 95° C.,1 minute annealing at 52° C. and 1.5 minute extension at 72° C.,followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (LifeTechnologies, Gaithersburg, Md.).

The results showed that the zamp1 gene maps 33.5 cR_(—)3000 from the topof the human chromosome 8 linkage group on the WICGR radiation hybridmap. Proximal and distal framework markers were CHLC.GATA62D10 andWI-3823 (D8S1511), respectively. The use of surrounding markerspositions the zamp1 gene in the 8p23.3–p23.2 region on the integratedLDB chromosome 8 map (The Genetic Location Database, University ofSouthhampton, WWW server located on the Internet).

EXAMPLE 4 Identification of DNA Encoding Full Length Zamp1 Polypeptideand Sequencing Thereof

The 5′ end of zamp1 coding sequence was obtained by PCR usingGenomeWalker® reagents (Clontech) in combination with a zamp1polypeptide-specific antisense primer ZC15591 (SEQ ID NO: 12) and thenconducting nested PCR with ZC15589 (SEQ ID NO: 13), according tomanufacturer's instructions, with the exception that 64° C. was used inthe primary reaction instead of the suggested 67° C. PCR products wererun on a 2% agarose gel (Gibco), and gel purified using Qiaex IIreagents (Qiagen) according to manufacturer's instructions. Productswere sequenced using ZC15589 (SEQ ID NO: 13) as a sequencing primer,revealing the extension of zamp1 polypeptide-encoding to a putativeinitiation methionine (nucleotides 1–201 or 1–219 of SEQ ID NO:9) andabout 250 base pairs of 5′ untranslated sequence.

EXAMPLE 5 Synthesis of Zamp1

A 45 amino acid residue zamp1 peptide (residues 23 to 67 of SEQ IDNO:10) was synthesized by solid phase peptide synthesis using a model431A Peptide Synthesizer (Applied Biosystems/Perkin Elmer, Foster City,Calif.). Fmoc-Lysine(Boc) resin (0.52 mmol/g; Anaspec Inc., San Jose,Calif.) was used as the initial support resin. 1 mmol Amino acidcartridges (Anaspec Inc., San Jose, Calif. and Applied Biosystems/PerkinElmer, Foster City, Calif.) were used for synthesis.2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyuroniumhexafluorophosphate(HBTU), 1-Hydroxybenzotriazole (HOBt), 2 M N,N-Diisopropylethylamine,N-Methylpyrrolidone, Dichloromethane (all from Applied Biosystems/PerkinElmer, Foster City, Calif.), along with piperidine (Aldrich ChemicalCo., St. Louis, Mo.) and 0.5 M acetic anhydride capping solution(Advanced ChemTech, Louisville, Ky.), were used as synthesis reagents.

The Peptide Companion software (Peptides International, Louisville, Ky.)was used to help predict the aggregation potential for the synthesis forzamp1. Synthesis was performed using both single and double couplingcycles. Also, acetylation was used where difficult couplings werepredicted.

The peptide was cleaved from the solid phase by the standard TFAcleavage procedure (according to Peptide Cleavage manual, AppliedBiosystems/Perkin Elmer). Purification of the peptide was done byRP-HPLC using a C18, 52 mm×250 mm, preparative column (Vydac, Hesperia,Calif.). Fractions from the column were collected and analyzed for thecorrect mass by electrospray mass spectrometry; the purity was analyzedby analytical RP-HPLC, using a C18, 4.6 mm×250 mm column (Vydac,Hesperia, Calif.). The mass spectrometry analysis confirmed the desiredmolecular weight of the reduced form of zamp1, i.e., 5158. Purifiedfractions were frozen and then lyophilized.

The reduced peptide was dissolved in 6 M guanidine HCl (Aldrich ChemicalCo.) at an initial concentration of 2 mg/ml. This solution was thenadded slowly to 2.1 volume equivalents of 1 M guanidine HCl along with0.52 volume equivalents of DMSO (Aldrich Chemical Co.). The oxidationwas monitored with analytical RP-HPLC using the same analytical C18column; the oxidation was complete at 48 hours. Salts were removed fromthe reaction mixture using solid phase extraction C18 cartridges(Waters, Milford, Mass.). The eluant containing the oxidized peptide isconcentrated and then purified using RP-HPLC semi-prep C18 column(Vydac, Hesperia, Calif.). Four distinct peaks were determined tocorrespond to the fully oxidized form of zamp1 by electrospray LCMS. Thepeak referred to as peak 2, as it was the second peak to elude byRP-HPLC, was found to contain the conserved defensin disulfide pattern,by a process of elimination using partial digest and peptide mapping ofall four peaks. This peak was isolated, frozen and lyophilized.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polynucleotide molecule encoding a protein, wherein theencoded protein comprises an amino acid sequence having at least 95percent sequence identity with amino acid residues 23 to 67 of SEQ IDNO:10, and wherein the encoded protein has antimicrobial activity. 2.The isolated polynucleotide molecule of claim 1 wherein the encodedprotein comprises amino acid residues selected from the group of 22 to67 of SEQ ID NO:10, 21 to 67 of SEQ ID NO:10, 20 to 67 of SEQ ID NO:10,and 1 to 67 of SEQ ID NO:10.
 3. The isolated polynucleotide molecule ofclaim 1 wherein the encoded protein has three disulfide bonds.
 4. Theisolated polynucleotide molecule of claim 1 wherein the amino acidpercent identity is determined using a FASTA program with ktup=1, gapopening penalty=10, gap extension penalty=1, and substitutionmatrix=BLOSUM62, with other parameters set as default.
 5. The isolatedpolynucleotide molecule of claim 1 wherein the differences between theencoded protein and amino acid residues 23 to 67 of SEQ ID NO:10 are dueto conservative amino acid substitutions.
 6. The isolated polynucleotidemolecule of claim 1 wherein the encoded protein has antimicrobialactivity to at least one microbe selected from the group of bacteria,anaerobic organisms, protozoans, fungi, and viruses.
 7. The isolatedpolynucleotide molecule of claim 1 wherein the encoded protein hasantimicrobial activity to bacteria.
 8. The isolated polynucleotidemolecule of claim 7 wherein the bacteria are gram positive.
 9. Theisolated polynucleotide molecule of claim 7 wherein the bacteria aregram negative.
 10. An expression vector comprising the followingoperably linked element: a transcription promoter; a DNA segmentcomprising the isolated polynucleotide molecule encoding a protein ofclaim 1; and a transcription terminator.
 11. The expression vector ofclaim 10 wherein the DNA segment further encodes a secretory signalsequence operably linked to the protein.
 12. A cultured cell into whichhas been introduced an expression vector of claim 10, wherein the cellexpresses the protein encoded by the DNA segment.
 13. A method ofproducing a protein comprising: culturing a cell into which has beenintroduced an expression vector of claim 10, whereby the cell expressesthe protein encoded by the DNA segment; and recovering the expressedprotein.
 14. An isolated polynucleotide molecule encoding a polypeptidecomprising amino acid residues 23 to 67 of SEQ ID NO:10.